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WO1998049345A1 - Procedes et compositions utiles pour l'affichage differentiel d'adn cibles - Google Patents

Procedes et compositions utiles pour l'affichage differentiel d'adn cibles Download PDF

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Publication number
WO1998049345A1
WO1998049345A1 PCT/US1998/008616 US9808616W WO9849345A1 WO 1998049345 A1 WO1998049345 A1 WO 1998049345A1 US 9808616 W US9808616 W US 9808616W WO 9849345 A1 WO9849345 A1 WO 9849345A1
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pcr
primer
repeat
sequence
sequences
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PCT/US1998/008616
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English (en)
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Cassandra L. Smith
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Trustees Of Boston University
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Priority to AU72652/98A priority Critical patent/AU7265298A/en
Publication of WO1998049345A1 publication Critical patent/WO1998049345A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention relates to the identification of expressed genes, and in particular, methods and compositions for distinguishing between the expression of genes in two or more biological samples.
  • a subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one sample and not present in another (e.g., present in a particular species, tissue or cell and not present in another species, tissue or cell). See generally, Current Protocols in Molecular Biology, Section 5.8.9 (1990). In the protocol, cDNA containing the gene(s) of interest ["+cDNA”] is prepared with EcoRI ends and the cDNA not containing the gene(s) of interest ["-cDNA”] is prepared with blunt ends.
  • the +cDNA is mixed with a 50-fold excess of -cDNA inserts and the mixture is heated to make the DNA single-stranded. Thereafter, the mixture is cooled to allow for hybridization. Annealed cDNA inserts are ligated to a vector and transfected.
  • the only +cDNA likely to be double-stranded with an ⁇ coRI site at each end are those not hybridized to something in the -cDNA preparation; in other words, where a complementary sequence is in the -cDNA preparation, the sequence will not be transfected.
  • sequences unique to the +cDNA preparation will be cloned and amplified.
  • DDRT-PCR differential display of mRNAs using arbitrarily primed polymerase chain reaction
  • the polymerase chain reaction is described by Mullis et al. in U.S. Patents Nos. 4,683,195, 4,683,202 and 4,965,188, hereby incorporated by reference.
  • the PCR process consists of introducing a molar excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence.
  • the two primers are complementary to their respective strands of the double-stranded sequence.
  • the mixture is denatured and then allowed to hybridize.
  • the primers are extended with a thermostable DNA polymerase so as to form complementary strands.
  • the steps of denaturation, hybridization, and polymerase extension can be repeated as often as needed to obtain a relatively high concentration of a segment of the desired target sequence.
  • the target is mRNA; the mRNA is, however, treated with reverse transcriptase in the presence of oligo(dT) primers to make cDNA prior to the PCR process.
  • the PCR is carried out with random primers in combination with the oligo(dT) primer used for cDNA synthesis.
  • the amplified products are placed in side-by-side lanes of a gel; following electrophoresis, the products can be compared or "differentially displayed.”
  • DDRT-PCR while an improvement over subtractive hybridization, has a number of drawbacks.
  • the use of arbitrary random primers can cause faint banding at essentially every position of the gel.
  • the process is generally biased toward high-copy number genes.
  • E. Haag et al, Biotechniques 17:226-228 (1994) describes an improved DDRT-PCR method, whereby the use of the standard oligo-dT primer in the PCR step is omitted to decrease the faint banding at essentially every position of the electrophoresis gel.
  • a second arbitrary primer was utilized in PCR.
  • O.C Another example is O.C.
  • the present invention relates to the identification of expressed genes, and in particular, methods and compositions for distinguishing between the expression of genes in two or more biological samples.
  • the present invention employs oligonucleotide primers targeting CAG repeats. Such repeats are known to be important in an increasing number of neurological diseases.
  • the present invention contemplates first and second oligonucleotide primers, said first oligonucleotide primer comprising a CTG repeat and said second oligonucleotide primer comprising a CAG-repeat.
  • the primer containing a CTG repeat is contemplated for amplifying unique sequences flanking the 5 '-side of the CAG repeat sequence in the target.
  • the primer containing the CAG repeat is contemplated for amplifying the 3' -flanking sequences.
  • oligonucleotide comprising (CAG) n or (CTG) n is contemplated, wherein n is a whole number between 1 and 15, and more preferably, between 2 and 12, and still more preferably, between 6 and 12.
  • oligonucleotides comprising the general formula X p (CAG) n X p or X p (CTG) n X p are contemplated, wherein X is selected from the group consisting of A,T,C or G and p is a whole number between 1 and 15, and n is a whole number between 2 and 12, and still more preferably, between 6 and 12.
  • X is selected from the group consisting of A,T,C or G and p is a whole number between 1 and 15, and n is a whole number between 2 and 12, and still more preferably, between 6 and 12.
  • CAG and CTG repeats as other repeating and non-repeating nucleotide sequences will find use as primers with the present invention.
  • sample and “specimen” in the present specification and claims are used in their broadest sense. On the one hand they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, cells as well as solid tissue (including both normal and diseased tissue). These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms. It is also not intended that the invention be limited by the particular purpose for carrying out the biological reactions. In one medical diagnostic application, it may be desirable to differentiate between normal and diseased tissue.
  • CSF cerebrospinal fluid
  • the present invention contemplates a method of analyzing nucleic acid in a sample, comprising: a) providing: i) a sample containing nucleic acid, ii) a first oligonucleotide primer comprising a CTG repeat, iii) a second oligonucleotide primer comprising a CAG-repeat, and iv) a polymerase and PCR reagents; b) preparing said nucleic acid from said sample under conditions so as to produce amplifiable nucleic acid; c) amplifying said nucleic acid with said first and second primers, said polymerase and said PCR reagents under conditions such that amplified product is generated; d) detecting said amplified product.
  • adapter oligonucleotides are employed.
  • PCR-suppression is employed in conjunction with the primers described above. It is not intended that the present invention be limited by the means of detection. In one embodiment, said detecting comprises gel electrophoresis. Furthermore, it is not intended that the present invention be limited to the use of PCR amplification as a means of characterizing the samples. In some embodiments, the present invention completates targeting methods to detect differences in nucleic acid samples, whereby the sample are identified thought the use of DNA arrays (See e.g., the methods of Chee et al., Science 274, 610 [1996]; DeRisi et al., Nat. Genet.
  • the present invention can be used with particular success when comparing samples.
  • the present invention contemplates amethod of analyzing expressed genes in biological samples.
  • Clinical samples are specifically contemplated within the scope of the present invention.
  • the present invention contemplates the primers of the present invention as unique compositions.
  • the present invention also contemplates kits containing these novel compositions.
  • the kit comprises a first primer comprising a CTG repeat and said second oligonucleotide primer comprising a CAG-repeat.
  • nucleic acid sequence and “nucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • nucleic acid sequence and “nucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • complementary or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules.
  • the sequence "C-A- G-T,” is complementary to the sequence "G-T-C-A.”
  • Complementarity can be “partial” or “total.”
  • Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules.
  • Total or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
  • nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity).
  • a nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding the probe will not hybridize to the second non-complementary target.
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 »H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 »H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent 50X Denhardt's contains per
  • low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
  • conditions which promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • substantially homologous refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • the term “substantially homologous” refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • substantially homologous refers to any probe which can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex.
  • Hybridization and the strength of hybridization is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R ⁇ t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support [e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
  • a solid support e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency” typically occurs in a range from about T m -5°C (5°C below the T m of the probe) to about 20°C to 25°C below T m .
  • a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
  • amplifiable nucleic acid is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that
  • amplifiable nucleic acid will usually comprise “sample template.”
  • sample template refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest.
  • background template is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
  • any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • PCR reagents or "PCR materials”, which herein are defined as all reagents necessary to carry out amplification except the polymerase, primers and template.
  • PCR reagents nomally include nucleic acid precursors (dCTP, dTTP etc.) and buffer.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i. e. , in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • the term "probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest.
  • a probe may be single- stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labelled with any
  • reporter molecule so that it is detectable using any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
  • enzyme e.g., ELISA, as well as enzyme-based histochemical assays
  • fluorescent, radioactive, and luminescent systems It is not intended that the present invention be limited to any particular detection system or label.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring.
  • an end of an oligonucleotide is referred to as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
  • discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.
  • the promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • isolated when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as
  • purified or “to purify” refers to the removal of undesired components from a sample.
  • substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • An "isolated polynucleotide” is therefore a substantially purified polynucleotide.
  • the term "gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
  • genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
  • sample as used herein is used in its broadest sense and includes environmental and biological samples.
  • Environmental samples include material from the environment such as soil and water.
  • Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables).
  • Figure 1 is a display of fluorescein-labeled Mse I CAG-containing DNA fragments from three pairs of monozygotic twins.
  • Genomic DNAs were digested with the restriction enzyme Mse I, ligated to adapter oligonucleotides of known sequence and hybridized to an immobilized single-stranded probe containing (CAG) 12 repeats.
  • Captured DNAs were amplified by PCR with fluorescein-labeled primer 3 (Table 1 ) and displayed on an ALF sequencing instrument. DNAs were from three pairs of monozygotic twins (pair 1. lanes 15 and 16; pair 2, lanes 17 and 18; pair 3, lanes 19 and 20).
  • FIG. 1 shows HD-sequence information used in experiments.
  • a partial sequence of the first exon of the HD gene (Genbank accession no. L34020) is shown along with the locations of HD primers (oligonucleotides 9-12; Table 1 ; horizontal arrows) and Sau96 I restriction sites (vertical arrows) surrounding a CAG repeat (bold).
  • Figure 3 is a display of different HD alleles from three related individuals (lanes 1 , HD-A; 2, HD-B; and 3, HD-C) and an anonymous unrelated control (lane 4) in a Sau96 I
  • CAG repeat-containing primer 14 and fluorescein-labeled primer 5 (Table 1). This experiment displays Sau96 I CAG-containing genome subsets en masse. The differentially displayed fragments eluting at -227 min hybridized to an HD-specific capture probe (data not shown). Also not shown are the results of experiments using adapter primers only. These experiments detected the same differentially displayed fragments eluting at -227 min as shown in (C).one embodiment of the primers of the present invention (a "K primer”) partially hybridized to one strand of a denatured double-stranded template.
  • Figure 4 is a schematic showing the PCR-based genomic differential display method used to amplify interspersed repeats and flanking unique sequences.
  • Figure 5 is a genomic differential display targeting different (CAG) n -containing genome subsets.
  • DNA sample was amplified first with a fluorescein-labeled T-primer and an A-primer (oligonucleotides 11 and 3, respectively, Table 2). Then, a second PCR amplification was done using the same fluorescein-labeled T-primer (oligonucleotide 11 , Table 2) and various A-primers; oligonucleotide 5 (lane 1), 9 (lane 2), 8 (lane 3), or 6 (lane 4) (Table 2). No PCR products were generated if fluorescein-labeled T-primer
  • oligonucleotide 11, Table 2 was used alone (lane 5). Fluorescence intensity versus fragment size is shown. Each lane is independently auto-scaled.
  • Figure 6 shows a comparison of CAG repeat-containing genomic subsets from monozygotic twins amplified by PCR using 3 '-(A) or 5 '-terminally anchored (B) T-repeat primers.
  • DNA from two pairs of monozygotic twins was digested with Hae III, ligated to adapter (oligonucleotides 1 and 2, Table 2) and PCR amplified. The products were fractionated by size on an ALF sequencing instrument.
  • A Both PCR amplifications used a 3 '-terminally anchored fluorescein-labeled T-primer (oligonucleotide 12, Table 2).
  • the A- primers were oligonucleotides 3 and 8 (Table 2) in the first and second PCR reactions, respectively.
  • Both PCR amplifications used a 5 '-anchored fluorescein-labeled T-primer (oligonucleotide 11, Table 2).
  • the A-primers were oligonucleotides 3 and 8 in the first and second PCR, respectively (Table 2).
  • the present invention relates to the identification of expressed genes, and in particular, methods and compositions for distinguishing between the expression of genes in two or more biological samples.
  • the present invention contemplates methods and compositions for differential display with genomic DNA, cDNA, and other nucleic acid species.
  • genomic DNA genome complexity is reduced by the methods of the present invention, and focus is provided by targeting genome subsets containing specific interspersed repeats.
  • the targeted genomic subsets contain a CAG trinucleotide repeating sequence. Using primers descried to such repeats, surrounding unique sequences are identified.
  • interspersed repeat-containing fragments can be targeted in the same manner, e.g., fragments containing SINEs, LINEs, LTRs (long terminal repeats), sequences coding for particular protein motifs or ' s-acting sequence elements.
  • SINEs e.g., SINEs, LINEs, LTRs (long terminal repeats), sequences coding for particular protein motifs or ' s-acting sequence elements.
  • Differential display of cDNAs could also be enhanced by a similar use of interspersed repeats to target interesting cDNA subsets.
  • the display method of the present invention will detect polymorphisms that may arise in the unique sequences surrounding the repeat. However, each displayed polymorphism must be characterized individually in order to understand its origin.
  • the informativeness of these and other differential display methods will be maximized when large amounts of data can be analyzed automatically, such as by high throughput automated analysis using signal processing methods.
  • the display method can be readily applied to assess the variation within monozygotic twin pairs. At a minimum, such experiments should provide quantitative information on genome stability, and they have the potential to reveal interesting facets of twin biology.
  • the methods and compositions of the present invention provide general means of targeting a variety of DNA sources to allow comparison of samples, to target samples to reduce complexity, to focus analysis on DNA fragments containing specifici sequences, and to target rare nucleic acid species.
  • One skilled in the art will recognize many applications of the methods and compositions of the present invention.
  • the method can be generalized to amplify genome subsets containing a variety of targets including various consensus sequences coding for cis-acting elements or protein motifs.
  • targets including various consensus sequences coding for cis-acting elements or protein motifs.
  • the methods of the present invention use a sequence specific capture step and PCR amplicification to focus analysis on genome subsets containing target sequences.
  • Adaptor-tagged genomic restriction fragments i.e., restriction fragments ligated to known oligonucleotides
  • the captured fragments are amplified by
  • PCR using primers to the adaptor sequences alone, or in the presence of a primer complementary to the targeted sequence.
  • the amplified and labeled gragments can then be characterized on a sequencing gel or other means known in the art.
  • the method allows the isolation and analysis of genome subsets containing targeted repeat sequences.
  • the method allows the isolation and analysis of genome subsets containing targeted repeat sequences.
  • the method takes advantage of PCR-suppression ["PS;" Siebert et al., Nucleic Acids Res. 23:1087-1088 (1995); Lukyanov et al., Anal. Biochem. 229:198-202 (1995)] to amplify targeted repeat-containing sequences along with a unique flanking sequence directly from genomic DNA.
  • PS was used to selectively amplify genomic DNA sequences adjacent to known sequences and to improve subtractive hybridization protocols [Lukyanov et al., supra; Diachenko et al., Proc. Natl. Acad. Sci. USA 93:6025-6030 (1996)].
  • the outline of a PCR-based genomic differential display method is shown in Figure 4.
  • the template is genomic DNA digested with a restriction enzyme and ligated to known adapter sequences.
  • a 40-base adapter sequence is used to promote the annealing of the fragment ends to each other.
  • the annealed complementary ends suppress annealing or extension of shorter PCR primers (A-primers) complementary to the same sequences.
  • PS for PCR suppression [Siebert et al. (1995); Lukyanov et al. (1995); Diachenko et al. (1996)].
  • the A-primer corresponds to the self-complementary end adapter sequences.
  • the second primer termed the T-primer, is complementary to a genomic target sequence located in the single-stranded section of the end-annealed genomic fragments.
  • Single-stranded PCR products produced by extension of annealed T-primers, no longer have complementary ends and will not be subjected to PS.
  • Genomic fragments that do not contain the target sequence will remain end-annealed and cannot be extended by the A-primer. Occasional extension of an annealed A-primer to the original template will produce a single-stranded fragment that is still subject to PS because of its complementary ends.
  • PS ensures that only fragments containing the targeted sequence are efficiently amplified by PCR.
  • the T-primers used here target CAG-repeat sequences.
  • a T-primer containing a CTG-repeat amplifies unique sequence flanking the 5 '-side of the CAG repeat sequence, whereas the 3 '-flanking sequence is amplified with the T-primer containing a CAG-repeat sequence.
  • the nucleic acid content of cells consists of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA contains the genetic blueprint of the cell.
  • RNA is involved as an intermediary in the production of proteins based on the DNA sequence. RNA exists in three forms within cells, structural RNA (i.e., ribosomal RNA "rRNA”), transfer RNA ("tRNA”), which is involved in translation, and messenger RNA ("mRNA"). Since the mRNA is the intermediate molecule between the genetic information encoded in the DNA, and the corresponding proteins, the cell's mRNA component at any given time is representative of the physiological state of the cell. In order to study and utilize the molecular biology of the cell, it is therefore important to be able to purify mRNA, including purifying mRNA from the total nucleic acid of a sample.
  • RNA is complicated by the presence of ribonucleases that degrade RNA (e.g., T. Maniatis et al, Molecular Cloning, pp. 188-190, Cold Spring Harbor Laboratory [1982]). Furthermore, the preparation of amplifiable RNA is made difficult by the presence of ribonucleoproteins in association with RNA. (See, R. J. Slater, In: Techniques in Molecular Biology, J.M. Walker and W. Gaastra, eds., Macmillan, NY, pp. 113-120 [1983]).
  • the steps involved in purification of nucleic acid from cells include 1) cell lysis; 2) inactivation of cellular nucleases; and 3) separation of the desired nucleic acid from the cellular debris and other nucleic acid.
  • Cell lysis may be achieved through various methods, including enzymatic, detergent or chaotropic agent treatment.
  • Inactivation of cellular nucleases may be achieved by the use of proteases and/or the use of strong denaturing agents.
  • separation of the desired nucleic acid is typically achieved by extraction of the nucleic acid with phenol or phenol-chloroform; this method partitions the sample into an aqueous phase (which contains the nucleic acids) and an organic phase (which contains other cellular components, including proteins).
  • the structure of the mRNA molecule may be used to assist in the purification of mRNA from DNA and other RNA molecules.
  • poly-A tail or "poly-A track”
  • one means of isolating RNA from cells has been based on binding the poly-A tail with its complementary sequence (i.e., oligo-dT), that has been linked to a support such as cellulose.
  • oligo-dT its complementary sequence
  • the hybridized mRNA/oligo-dT is separated from the other components present in the sample through centrifugation or, in the case of magnetic formats, exposure to a magnetic field.
  • the mRNA is usually removed from the oligo-dT. However, for some applications, the mRNA may remain bound to the oligo-dT that is linked to a solid support.
  • RNA Ribonucleic acid
  • mammalian e.g., liver tissue
  • the present invention contemplates the isolation of PolyA+ RNA from extracts, including direct isolation from crude extracts.
  • Nonphosphorylated oligonucleotides were from Operon Technologies (Alameda, CA). DNA samples were from an anonymous healthy donor, a Huntington's disease (HD)-affected kindred and monozygotic twins.
  • HD Huntington's disease
  • Genomic DNA (100 ng), digested with the restriction enzyme Sau3A I or Mse I, was ligated with 50 pmoles of corresponding adapters (oligonucleotides 1 and 2, or and 3 and 4 for Mse I, respectively; Table 1) in a 10-20 ⁇ l total reaction volume overnight at 14°C with 40 units of T4 DNA ligase (New England Biolabs). Each pair of oligonucleotides was first annealed by cooling the mixture from 70°C to 10°C in a 1-hr period.
  • Ligase was inactivated by heating at 75°C for 10 min, and a fill-in reaction was done at 72°C for 10 min after the addition of dNTPs (100 ⁇ M each) and 0.5 units of AmpliTaq DNA polymerase (Perkin Elmer). DNA was phenol extracted, precipitated with ethanol. washed with 70% ethanol, dried and dissolved in TE buffer (10 mM Tris-HCl, pH 8.0/1 mM EDTA).
  • a biotinylated oligonucleotide (10 pmol) containing a (CAG) 12 or (CCG) 12 sequence (oligonucleotide 7 or 8, respectively; Table 1) was mixed with 50 ng of ligation products in 50 ⁇ l of TE buffer containing 2 ⁇ M of the corresponding adapter oligonucleotides to prevent annealing of the fragment ends to each other.
  • the sample was heated to 95°C, slowly cooled to room temperature, added to 100 ⁇ g of prewashed streptavidin-coated magnetic beads M-280 [as directed by Dynal (Oslo, Norway)] /using a 3-fold molar excess of biotin-binding capacity over biotinylated oligonucleotides/ and incubated at room temperature for 1 hr with gentle rotation.
  • the beads were collected with a magnet, washed twice at 55- 60°C for 20 min with 3x SSC (lx SSC: 0.15 M NaCl/ 15 mM sodium citrate)/ 0.5% SDS, and, at room temperature, twice each, with TE containing 1 M NaCl and with TE alone. Beads with captured DNA were stored in TE buffer at 4°C.
  • One-fifth of the captured DNA was amplified by PCR in a PTC- 100 thermal cycler (MJ Research, Inc.) as described in Lisitzyn et al, Science 259:946-951(1993).
  • the 50- ⁇ l reaction contained 67 mM Tris-HCl, pH 8.8/ 4 mM MgCl 2 / 16 mM (NH 4 ) 2 S0 4 / 10 mM 2- mercaptoethanol/ 300 ⁇ M of each dNTP/ 2 units of AmpliTaq DNA polymerase/ 5 ⁇ M fluorescein-labeled adapter primer (i. e., in the absence of a repeat primer).
  • the samples were incubated at 94°C for 3 min and subjected to 20-23 cycles, each consisting of 1 min at 94°C and 3 min at 72°C, and a final incubation at 72°C for 5 min (e.g., Fig. 1).
  • captured DNA was amplified by PCR using the appropriate adapter primer (2.5 ⁇ M) and a primer complementary to the repeat (5 ⁇ M, oligonucleotides 13 or 14; Table 1). PCR conditions were as described above, except that the annealing temperature was 45°C. The primer labeled with fluorescein varied in different experiments.
  • Amplified PCR products (1-2 ⁇ l) were denatured for 5 min at 90°C in 4 ⁇ l of a stop solution containing 6 mg/ml of dextran blue and 0.1% SDS in deionized formamide. loaded onto a 6% denaturing polyacrylamide gel and analyzed on an ALF DNA Sequencer (Pharmacia-Biotech., Sweden). The results were displayed using Fragment Manager software provided with the instrument.
  • the size standard was a fluorescein-labeled 100-base pair (bp) ladder (Gibco-BRL). The electrophoresis conditions fractionated fragments from 80 to 800 bp.
  • CAG-containing double-stranded fragments obtained after capture and PCR amplification with adapter primers were cloned using a TA cloning kit (Invitrogen. San Diego, CA). Randomly chosen clones were sequenced using a Sequenase 2.0 kit (Pharmacia- Biotech., Sweden) and an ALF sequencer.
  • Each 50- ⁇ l reaction contained 100 ng DNA/ 0.5 ⁇ M of each primer/ 20 mM Tris-HCl, pH 8.4/ 50 mM KC1/ 200 ⁇ M dNTPs/ 2 mM MgCl 2 / 3.5% formamide/ 15% glycerol/2.5 units of AmpliTaq DNA polymerase. Cycling conditions were 94°C for 3 min, followed by 35 cycles at 94°C for 1 min, 64°C for 1 min, and 72°C for
  • HD-fragments obtained from PCR amplification of genomic DNA with primers 11 and 12 (Table 1), were digested with Sau96 I and ligated to Sau9 ⁇ I adapters (oligonucleotides 5 and 6; Table 1). The resulting HD- fragments were then amplified by PCR using adapter primer 5 and fluorescein-labeled CTG- containing repeat primer 13 (Table 1). PCR conditions and analysis were as described above.
  • CAG repeat-containing genomic subsets captured from Sau9 ⁇ I-digested and tagged DNAs, were amplified by PCR using fluorescein-labeled primer 5 and a CAG-containing repeat primer (primer 14) or CTG-containing repeat primer 13 (Table 1). Two- ⁇ l aliquots were displayed as described above. The remaining PCR product (-100 ng) was hybridized overnight at 37°C to an immobilized HD-specific capture probe in 6x SSC/ 5x Denhardt's solution/ 0.5%) SDS/ 100 ⁇ g/ml of herring sperm DNA/ 100 pmol each of oligonucleotides (CAG) 6 and (CTG) 6 .
  • CAG oligonucleotides
  • the capture probe generated by PCR using oligonucleotide 10 and biotinylated oligonucleotide 11 (Table 1), was a 173-bp fragment upstream from the CAG repeat in the first exon of the HD gene.
  • the gel-purified PCR product (175 ng, 1.5 pmol) was immobilized on streptavidin-coated magnetic beads and treated with alkali, as recommended by the manufacturer, Dynal. After hybridization, the beads were washed once in lx SSC/ 0.5% SDS at 65°C for 2 firs and rinsed twice with TE at room temperature. Captured fragments were released from the beads by boiling for 5 min in stop solution and displayed as described above.
  • CAG- (and CGG-) trinucleotide repeating sequences were targeted because of the importance of these repeats in neurodegenerative diseases [Ross et al. (1993) Trends Neurosci. 16:254-260; Sutherland & Richards (1995) Proc. Natl. Acad. Sci. USA 92:3636-3641]. It should be noted that only ten capture probes are needed to profile all trinucleotide repeat sequences.
  • genomic DNA is cleaved with a restriction enzyme which cuts outside of the targeted repeat sequence.
  • the restriction fragments are tagged at their ends by ligation to adapters, i. e., oligonucleotides of known sequence.
  • the adapters permit subsequent amplification and labeling of the fragments by PCR.
  • the fragments are denatured, hybridized to a biotinylated single- stranded oligonucleotide probe containing a sequence, (CAG), 2 [or (CCG) 12 ], complementary to the targeted repeat sequence, and captured on streptavidin-coated magnetic beads. Captured fragments are then amplified by PCR using an adapter primer alone or in combination with a primer complementary to the targeted repeat.
  • Captured fragments containing CAG- (or CCG-) repeating sequences were amplified by PCR using an adapter primer and cloned. The clones were hybridized to the targeted repeat sequence. As expected, most (-90% and -60% of the putative CAG and CCG clones, respectively) hybridized to the corresponding probes. Sequencing of four randomly-selected
  • CAG-containing clones revealed the presence of four different CAG repeated sequences, /. e. , (CAG) 3 , (CAG) 4 , (CAG) 6 CCAGAGCCAG, and (CAG) 2 ACAGCA. These results showed that the capture procedure generated genome subsets enriched for targeted repeat-containing sequences.
  • the completeness of the capture CAG-containing genome subset was evaluated by determining the total number of 32 P end-labeled Hind III restriction fragments captured by hybridization to an immobilized oligonucleotide containing (CAG) 12 (oligonucleotide 7; Table 1). A total of -0.5% of the fragments (or -5 x 10 3 fragments) were captured.
  • Fig. 1 Display of CAG repeat-containing fragments from three pairs of monozygotic twins is shown in Fig. 1.
  • genomic DNAs were digested with Mse I, ligated to an adapter consisting of oligonucleotides 3 and 4 (Table 1 ) and hybridized to an immobilized single-stranded oligonucleotide probe containing (CAG) ⁇ (oligonucleotide 7; Table 1).
  • CAG oligonucleotide 7; Table 1
  • the captured DNA was amplified by PCR using a fluorescein-labeled adapter primer (oligonucleotide 3; Table 1).
  • the fluorescence intensity versus elution time profiles shown in Fig. 1, represents size-fractioned fluorescein-labeled fragments.
  • Mse I genome subset is shown. Large fragments will not be efficiently amplified by PCR or, even if amplified, they may be outside the size range analyzed.
  • the annealing of the inverted terminal repeats at the ends of the fragments may prevent PCR amplification of some fragments [Siebert et al. (1995) Nucleic Acids Res 23:1087-1088; Lukyanov et al. (1995) Anal. Biochem. 229:198-202].
  • the hairpin structures formed by long CAG repeats [Mariappan et al. (1996) Nucleic Acids Res. 24:775-783; Gacy et al.
  • the genomic differential display method was tested for its ability to distinguish different HD alleles (Fig. 3).
  • the HD sequence relevant to these experiments is shown in Fig. 2. Many differences were expected between the samples used in these experiments. Thus, a number of control experiments were used to identify the HD-containing fragments.
  • the length of the HD CAG repeat was determined in three members of an HD- affected kindred (HD-A, -B, and -C) and an unrelated (control) individual (Fig. 3A).
  • the control sample had two normal alleles, i. e., both alleles had - ⁇ 30 repeats, as did the HD-A sample.
  • Both the HD-B and HD-C samples had two expanded alleles ( 40 repeats); these HD homozygotes were reported previously [Wexler et al. (1987) Nature (London) 326: 194-197].
  • HD-B and HD-C samples also contained small amounts of fragments with shorter repeats. This type of HD mosaicism has been seen by others [Goldberg et al. (1993) Nature Genet. 5:174-179].
  • Fig. 3B tested the ability of the method to distinguish between normal and expanded HD alleles in the absence of other genomic restriction fragments.
  • the experiments took advantage of known Sau96 I restriction enzyme recognition sites located near the HD-CAG repeat sequence.
  • HD-specific PCR with primers oligonucleotides 11 and 12; Table 1 flanking the repeat was used to generate HD-containing fragments with Sau96 I recognition sites proximal to the repeat.
  • the PCR products were digested with Sau96 I, ligated to Sau96 I adapters (oligonucleotides 5 and 6; Table 1) and amplified by PCR using a S ⁇ w96 I adapter primer and a fluorescein-labeled repeat primer (oligonucleotides 5 and 13, respectively; Table 1). Clear differences were detected in the samples containing normal and expanded alleles (Fig. 3B).
  • Fig 3B was present in samples with normal HD alleles, but this fragment was absent in HD-B, and its amount was substantially reduced in HD-C.
  • An HD-containing fragment of about 130 bp long could contain 4-6 CAG repeats. This result suggests that during PCR variable length CAG repeats with a low number of repeats ( ⁇ 40) were converted to a constant length equal to that contained in the repeat primer [Weising et al. (1995) PCR Methods Apl. 4:249-255].
  • CAG repeat- containing fragments are known to form hairpin structures with stabilities that increase with repeat length [Mariappan et al. (1996) Nucleic Acids Res. 24:775-783; Gacy et al (1995) Cell 81 :533-540]. These structures appear to inhibit PCR amplification of expanded alleles as seen in these experiments and as reported before [Walsh et al. (1992) PCR Meth. Appl. 1:241-250; Demers et al. (1995) Nucleic Acids Res. 23:3050-3055].
  • the CAG-containing genome subsets shown in Fig. 3C were hybridized to the HD-specific capture probe and then displayed (data not shown). As expected, the fragments captured and displayed from the control and HD-A samples eluting at -227 min were not present in the HD- B and HD-C samples. Thus, the results in Fig. 3 show that our genomic differential display method is effective in distinguishing normal vs expanded CAG allele lengths.
  • genomic differential display using a two-step PCR protocol that targeted CAG-repeat containing genomic Hae III fragments is shown in Fig. 5.
  • genomic DNA digested with Hae III, and ligated to adapters (oligonucleotides 1 and 2, Table 2) was amplified in a two-step PCR protocol.
  • the A-primer was a 21 -base oligonucleotide 3 (Table 2) corresponding to the outermost part of the ligated adapter and the T-primer was a fluorescein-labeled oligonucleotide 11 (Table 2) composed of 3' CTG-repeating sequence plus two unique bases at the 5 '-end.
  • PCR primers with 5 '-anchors are not as selective as PCR primers and 3 '-anchors [Broude et ⁇ /.(1997); Ziefhiewicz et al. (1994)].
  • the products of the first PCR were diluted and used as templates in a second PCR.
  • the first and second PCRs used the same T-primers but different A-primers.
  • the A-primer in the second PCR reaction was a 26- or 27-base oligonucleotide containing a 5 '-terminal 22 base segment complementary to the innermost adapter sequence plus a 3 '-terminal tetranucleotide (CCTT) or pentanucleotide (CCTTA, CCTTG, or CCTTT) sequence.
  • the CC dinucleotide corresponded to the remainder of the Hae III recognition sequences.
  • the 3' terminal TT, TTA, TTG and TTT bases (lanes 1, 2, 3 and 4, respectively, Fig.
  • the dinucleotide and trinucleotide anchors should reduce the total genomic H ⁇ e III fragment complexity by, approximately, 16-fold and 64-fold, respectively.
  • PCR amplfication depended on the presence of both an A- (data not shown) and a T-primer, since no PCR products were detected when these primers were used alone (Fig. 5, lane 5).
  • PCR using an A-primer with a 2 base 3' anchor amplified several hundred fragments within the size range of -100 to -80 base pairs (Fig. 5, lane 1). Lengthening of the 3' terminal anchor reduced the number of amplified fragments (Fig. 5, lanes 2-4).
  • PCR amplification was done with a 3 '-terminal 3 base anchored A-primer and a 3 '-terminal 2 base anchored T-primer (e.g., oligonucleotides 8 and 12, respectively, Table 2).
  • PCR amplification was done using a 3 '-terminal 3 base anchored A-primer and 5 '-terminal 2 base anchored T-primer (oligonucleotides 8 and 11, respectively, Table 2). Clear differences were detected when DNAs from different twin pairs were compared.
  • CAG-containing subsets were obtained from genomic DNA templates isolated several times from the same blood sample. PCR amplifications were done with 5' terminally anchored T-primer (oligonucleotide 11, Table 2) and different A-primers. PCR products displayed on the same PAAG were compared using methods under development for automatic analysis of displayed genomic subsets (Graber et al. , unpublished results). In brief, peak to peak comparisons were tabulated using a conditioned signal function calculated from dividing individual signal peak differences by the individual peak means after high pass filtering (to remove slowly varying backgrounds), low pass filtering (to remove rapidly varying background), and overall temporal alignment (and scaling) and normalization.
  • the complexity of genome subsets analyzed by the method described here is modulated by the total number of occurrences of the targeted repeat sequence, the restriction fragment size distribution, the sensitivity of the restriction enzyme to methylation, and the location (5'- or 3 '-terminal), composition and length of the anchor sequences on the primers.
  • the use of anchored PCR primers allows for a controlled complexity reduction (Fig. 5 and 6) so that particular fragments of interest can be isolated from gels containing low complexity subsets.
  • Restriction fragment length polymorphisms may be due to polymorphisms in the repeat sequence or in the unique flanking sequences. Since the T-primer can be anchored with unique sequence at its 3'- or 5 '-end, sample comparisons can be focused solely on unique sequences flanking a targeted repeat sequence or on unique sequence flanking the repeat plus the repeat sequence itself, respectively.
  • Genomic DNA was isolated from blood lymphocytes from anonymous donors and monozygotic twin samples obtained from E. Fuller Torrey by a standard phenol-based extraction procedure.
  • genomic differential display isolated genomic DNA was digested with a restriction enzyme and ligated to oligonucleotides of known sequence as described [Broude et ⁇ /.(1997)]. Briefly, human genomic DNA (300 ng) was digested with 10 units of H ⁇ e III or Sau9 ⁇ I at 37 C overnight in a 100 ⁇ l total volume reaction. The recessed Sau96 I ends were filled in using AmpliTaq DNA polymerase (Perkin-Elmer).
  • DNA was precipitated by ethanol, dissolved in 20 ⁇ l of sterile water, and blunt-end ligated to an excess (2 ⁇ M) of each adapter oligonucleotide (oligonucleotides 1 and 2, Table 2) at 16 C overnight in a 30 ⁇ l final volume, containing 50 mM Tris- ⁇ Cl, p ⁇ 7.6/10 mM MgCl 2 /0.5 mM ATP/10 mM dithiotreitol/ and 5 units of T4 DNA ligase (Life Technologies, Gaitherburg, MD). This reaction was done with uneven adapter lengths to insure all the adapter oligonucleotides were ligated to the genomic fragments with the same polarity.
  • each adapter oligonucleotide oligonucleotides 1 and 2, Table 2
  • the ligation reactions produced genomic restriction fragments with 5' twenty-six base single-stranded overhangs.
  • the ligation was terminated by incubation at 75 C for 5 min. DNAs were then purified form excess primers by passing the samples through Wizard DNA purification columns (Promega, Madison, WI). DNA was eluted into 50 ⁇ l of sterile water.
  • the prepared DNA (3-5 ng) was amplified by PCR in 50 ⁇ l reaction volume in PCR buffer II (10 mM Tris- ⁇ Cl, p ⁇ 8.3/ 50 mM KCI) from Perkin-Elmer, plus 2.5 mM MgCl 2 ,
  • Hot start PCR was performed at 94 C by adding 0.2 ⁇ M each of the adapter-primer (A-primer) and fluorescein-labeled CTG-containing target-primers (T-primers), e.g., oligonucleotides 3 and 11 or 12, respectively (Table 2).
  • A-primer adapter-primer
  • T-primers fluorescein-labeled CTG-containing target-primers
  • PCR mixtures were subjected to 20 - 25 amplification cycles consisting of incubations at 94 C for 3 sec, 65 C for 20 sec and 72 C for 30 sec in the PTC- 100TM Temperature Cycler (MJ Res. Inc., MA).
  • the products of this first PCR were diluted 1000-fold and used as templates for a second PCR amplification.
  • T-primers in the first and second PCR were oligonucleotides 3 and one of the oligonucleotides designated 4-10, respectively (Table 2).
  • PCR products (1-2 ⁇ l) were denatured for 3 min at 90 C in a stop solution (Pharmicia- Biotech, Sweden) containing 6 mg/ml of dextran blue and 0.1% sodium dodecyl sulfate in deoinized formamide, loaded onto a 6% denaturing polyacrylamide gel (PAAG) and analyzed on the ALF DNA sequencing instrument (Pharmacia-Biotech, Sweden). The results were visualized using the Fragment Manager software provided with the instrument. Fluorescein- labeled 50 - 500 base pair (bp) ladder (Pharmicia-Biotech, Sweden) was used as a size marker.
  • bp base pair
  • PCR amplification was investigated by cloning and sequencing randomly chosen amplification products obtained from Sau96 I-digested DNA. Oligonucleotides 11 and 13 (Table 2) were used as T- and A-primers, respectively.
  • the PCR products from the second PCR amplification were cloned using a TA cloning kit (Invitrogen, San Diego, CA). Plasmid DNAs were isolated and sequenced using a Sequenase 2.0 kit (Pharmacia-Biotech, Sweden) and an ALF sequencing instrument.
  • Sequence 10 is a fragment of transcriptional activator hSNF2a gene (see below).
  • Clone 3 did not contain repeat sequence of the T-primer.
  • BLAST homology score is 4xe-28 to accession number X76572.
  • BLAST homology score is 5.6xe-72 to accession number X73969.
  • BLAST homology score is 8.4xe-27 to accession number D26155.

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Abstract

L'invention concerne des procédés et des compositions permettant de différencier l'expression de gènes dans deux ou davantage d'échantillons biologiques. Dans un mode de réalisation, un procédé d'affichage différentiel génomique employant une PCR est décrit. Le procédé emploie une suppression par PCR pour réduire la complexité du génome et pour amplifier des fragments génomiques contenant une séquence de répétition intercalée ciblée. Les produits de PCR peuvent être affichés selon leur taille pour produire une empreinte génomique, ou clonés pour produire une banque de fragments contenant la séquence de répétition ciblée et leurs séquences flanquantes. Les amorces et le procédé de la présente invention permettent l'identification précise de gènes exprimés de façon différente dans divers types de cellules.
PCT/US1998/008616 1997-04-29 1998-04-29 Procedes et compositions utiles pour l'affichage differentiel d'adn cibles WO1998049345A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003093501A2 (fr) * 2002-04-30 2003-11-13 University Of Bremen Methodes d'identification et d'isolement de sequences d'acides nucleiques specifiques
US7387876B2 (en) * 2004-02-27 2008-06-17 President And Fellows Of Harvard College Amplification of trace amounts of nucleic acids
US8263760B2 (en) 2008-02-08 2012-09-11 Prosensa Holding Bv Methods and means for treating DNA repeat instability associated genetic disorders
US8268962B2 (en) 2007-07-12 2012-09-18 Prosensa Technologies B.V. Molecules for targeting compounds to various selected organs or tissues
US8304398B2 (en) 2006-04-20 2012-11-06 Academisch Ziekenhuis Leiden Therapeutic intervention in a genetic disease in an individual by modifying expression of an aberrantly or abnormally expressed gene
US8361979B2 (en) 2006-05-19 2013-01-29 Academisch Ziekenhuis Leiden Means and method for inducing exon-skipping
US9139828B2 (en) 2008-05-14 2015-09-22 Prosensa Technologies B.V. Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means
US9243245B2 (en) 2007-10-26 2016-01-26 Academisch Ziekenhuis Leiden Means and methods for counteracting muscle disorders
US9890379B2 (en) 2006-08-11 2018-02-13 Biomarin Technologies B.V. Treatment of genetic disorders associated with DNA repeat instability
US9896687B2 (en) 2003-03-21 2018-02-20 Academisch Ziekenhuis Leiden Modulation of exon recognition in pre-mRNA by interfering with the secondary RNA structure
US10179912B2 (en) 2012-01-27 2019-01-15 Biomarin Technologies B.V. RNA modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy
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USRE48468E1 (en) 2007-10-26 2021-03-16 Biomarin Technologies B.V. Means and methods for counteracting muscle disorders

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5459037A (en) * 1993-11-12 1995-10-17 The Scripps Research Institute Method for simultaneous identification of differentially expressed mRNAs and measurement of relative concentrations
US5565340A (en) * 1995-01-27 1996-10-15 Clontech Laboratories, Inc. Method for suppressing DNA fragment amplification during PCR
US5580726A (en) * 1994-04-29 1996-12-03 Geron Corporation Method and Kit for enhanced differential display
US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR
WO1997017469A1 (fr) * 1995-11-09 1997-05-15 Dana Farber Cancer Institute Methode de prediction du risque de morbidite et de mortalite par cancer de la prostate
US5741645A (en) * 1993-06-29 1998-04-21 Regents Of The University Of Minnesota Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) * 1985-03-28 1990-11-27 Cetus Corp
US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR
US5741645A (en) * 1993-06-29 1998-04-21 Regents Of The University Of Minnesota Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis
US5459037A (en) * 1993-11-12 1995-10-17 The Scripps Research Institute Method for simultaneous identification of differentially expressed mRNAs and measurement of relative concentrations
US5580726A (en) * 1994-04-29 1996-12-03 Geron Corporation Method and Kit for enhanced differential display
US5565340A (en) * 1995-01-27 1996-10-15 Clontech Laboratories, Inc. Method for suppressing DNA fragment amplification during PCR
WO1997017469A1 (fr) * 1995-11-09 1997-05-15 Dana Farber Cancer Institute Methode de prediction du risque de morbidite et de mortalite par cancer de la prostate

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BROUDE N. E., ET AL.: "DIFFERENTIAL DISPLAY OF GENOME SUBSETS CONTAINING SPECIFIC INTERSPERSED REPEATS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 94., 1 April 1997 (1997-04-01), US, pages 4548 - 4553., XP002911916, ISSN: 0027-8424, DOI: 10.1073/pnas.94.9.4548 *
DIATCHENKO L., ET AL.: "SUPPRESSION SUBTRACTIVE HYBRIDIZATION: A METHOD FOR GENERATING DIFFERENTIALLY REGULATED OR TISSUE-SPECIFIC CDNA PROBES AND LIBRARIES.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93., 1 June 1996 (1996-06-01), US, pages 6025 - 6030., XP002911922, ISSN: 0027-8424, DOI: 10.1073/pnas.93.12.6025 *
LUKYANOV K. A., ET AL.: "INVERTED TERMINAL REPEATS PERMIT THE AVERAGE LENGTH OF AMPLIFIED DNA FRAGMENTS TO BE REGULATED DURING PREPARATION OF CDNA LIBRARIES BY POLYMERASE CHAIN REACTION.", ANALYTICAL BIOCHEMISTRY., ACADEMIC PRESS INC., NEW YORK., vol. 229., 1 January 1995 (1995-01-01), NEW YORK., pages 198 - 202., XP002911921, ISSN: 0003-2697, DOI: 10.1006/abio.1995.1402 *
MATZ M., ET AL.: "ORDERED DIFFERENTIAL DISPLAY: A SIMPLE METHOD FOR SYSTEMATIC COMPARISON OF GENE EXPRESSION PROFILES.", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 25., no. 12., 1 January 1997 (1997-01-01), GB, pages 2541/2542., XP002911919, ISSN: 0305-1048, DOI: 10.1093/nar/25.12.2541 *
SIEBERT P. D., ET AL.: "AN IMPROVED PCR METHOD FOR WALKING IN UNCLONED GENOMIC DNA.", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 23., no. 06., 1 January 1995 (1995-01-01), GB, pages 1087/1088., XP002911920, ISSN: 0305-1048 *
SUTHERLAND G. R., ET AL.: "SIMPLE TANDEM DNA REPEATS AND HUMAN GENETIC DISEASE.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 92., 1 April 1995 (1995-04-01), US, pages 3636 - 3641., XP002911917, ISSN: 0027-8424, DOI: 10.1073/pnas.92.9.3636 *
WEISING K., ET AL.: "GENOMIC FINGERPRINTING BY MICROSATELLITE-PRIMED PCR: A CRITICAL EVALUATION.", GENOME RESEARCH, COLD SPRING HARBOR LABORATORY PRESS, 1 January 1995 (1995-01-01), pages 249 - 255., XP002911918, ISSN: 1088-9051 *
ZIETKIEWICZ E., ET AL.: "GENOME FINGERPRINTING BY SIMPLE SEQUENCE REPEAT (SSR)-ANCHORED POLYMERASE CHAIN REACTION AMPLICIFICATION.", GENOMICS, ACADEMIC PRESS, SAN DIEGO., US, vol. 20., 1 January 1994 (1994-01-01), US, pages 176 - 183., XP002912035, ISSN: 0888-7543, DOI: 10.1006/geno.1994.1151 *

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